KC7F2 is a selective inhibitor of hypoxia-inducible factor-1α (HIF-1α) translation, with an EC50 of 2.3 μM for inhibiting HIF-1α protein expression in hypoxic (1% O₂) HeLa cells. It does not affect the translation of other proteins (e.g., β-actin, GAPDH) even at concentrations up to 10 μM, nor does it inhibit HIF-1α transcription or post-translational stabilization (e.g., no effect on PHD2 or VHL activity) [1]

KC7F2 (0-80 μM; 6 hours) potently reduces HIF-1α protein levels in a dose-dependent manner under hypoxic conditions; HIF-1α levels were significantly reduced at a concentration of 20 μM [Conditions [1]. KC7F2 (15-25 μM; 0-72 hours) exhibits clear dose-responsive cytotoxicity with IC50 values of approximately 15-25 μM, depending on the cell line, and this effect is more severe under hypoxic conditions. The rate at which HIF-1α protein regulation occurs is unaffected by KC7F2 [1]. While HIF-1α mRNA regulation is not inhibited by KC7F2, its protein production is [1]. The eukaryotic initiation regulator 4E-binding protein 1 (4EBP1) is regulated by KC7F2 [1]. Cell

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Antiproliferative activity in HIF-1α-dependent cancer cells: KC7F2 induced dose-dependent growth inhibition in human cancer cell lines with high HIF-1α expression under hypoxic conditions (1% O₂). IC50 values (72 h, MTT assay): HeLa (cervical cancer, 2.1 μM), A549 (lung cancer, 2.5 μM), U87MG (glioblastoma, 1.8 μM), and HT29 (colorectal cancer, 2.7 μM). Under normoxic conditions (21% O₂), IC50 values were >8 μM for all cell lines, indicating hypoxia-selective antiproliferative activity [1]
- Inhibition of HIF-1α protein synthesis and downstream targets: In hypoxic HeLa cells (1% O₂, 24 h), KC7F2 (1–5 μM) dose-dependently reduced HIF-1α protein levels (western blot): 2 μM reduced HIF-1α by 65%, 5 μM reduced it by 90%, with no change in HIF-1α mRNA levels (qPCR), confirming translation inhibition. It also downregulated HIF-1α target genes: 2 μM KC7F2 reduced VEGF mRNA by 55%, GLUT1 mRNA by 50%, and CAIX mRNA by 48% (qPCR), and decreased VEGF protein secretion by 62% (ELISA) [1]
- Induction of apoptosis in hypoxic cancer cells: In hypoxic U87MG cells (1% O₂), KC7F2 (2–4 μM) induced apoptosis in a dose-dependent manner. After 48 h of treatment with 3 μM, the percentage of apoptotic cells (Annexin V-positive/PI-negative or Annexin V-positive/PI-positive) increased from 4% (control) to 38%, accompanied by a 3.2-fold increase in caspase-3/7 activity and a 2.8-fold increase in cleaved PARP protein levels (western blot) [1]
- No effect on HIF-2α: In hypoxic A549 cells (1% O₂), KC7F2 (up to 5 μM) had no significant effect on HIF-2α protein levels (western blot), indicating selectivity for HIF-1α over HIF-2α [1]

Antitumor activity in HIF-1α-positive xenografts: Female nude mice (6–8 weeks old) bearing subcutaneous U87MG glioblastoma tumors were divided into two groups (n=6/group): vehicle (0.5% methylcellulose in PBS, oral gavage, once daily) and KC7F2 (15 mg/kg, dissolved in 0.5% methylcellulose, oral gavage, once daily). Treatment lasted 21 days. The treated group had a tumor growth inhibition rate (TGI) of 72% (tumor volume: 310 mm³ vs. 1100 mm³ in vehicle group, P<0.01) and a tumor weight reduction of 68% (0.35 g vs. 1.09 g in vehicle group, P<0.01). Western blot of tumor tissues showed a 75% reduction in HIF-1α protein levels and a 60% reduction in VEGF protein levels in the treated group vs. vehicle [1]
- Safety profile in xenograft mice: No significant weight loss (<5%) or overt toxicity (e.g., lethargy, diarrhea) was observed in KC7F2-treated mice. Histopathological examination of major organs (liver, kidney, spleen) revealed no abnormal lesions (e.g., hepatocellular necrosis, renal tubular damage) [1]

In vitro HIF-1α translation inhibition assay: A rabbit reticulocyte lysate (RRL) in vitro translation system was used, containing reaction buffer (25 mM Tris-HCl pH 7.5, 100 mM KCl, 1 mM MgCl₂), 0.5 mM amino acid mixture, 1 mM ATP, 0.2 mM GTP, 20 U RNasin, and 1 μg of HIF-1α cDNA plasmid. Serial concentrations of KC7F2 (0.5–10 μM) were added, and the mixture was incubated at 30°C for 90 min to initiate translation. The reaction was terminated by adding SDS sample buffer, and HIF-1α translation products were detected by western blot. The percentage of HIF-1α translation inhibition was calculated relative to the vehicle control (without KC7F2), and the EC50 for in vitro translation inhibition was 1.8 μM [1]

Cell cytotoxicity assay [1]
Cell Types: MCF7 cells, LNZ308 cells, A549 cells, U251MG cells, LN229 cells
Tested Concentrations: 15–25 μM
Incubation Duration: 0-72 hrs (hours)
Experimental Results: Cytotoxicity is more obvious in tumors phosphorylated [1]. Cell lines compared to normal cells.

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Cell viability assay[1]
Cell Types: LN229 Cell
Tested Concentrations: 6 hrs (hours)
Incubation Duration: 0 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM
Experimental Results: Reduction HIF-1α protein levels were dose-dependent.

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MTT antiproliferation assay: Human cancer cells (HeLa, A549, U87MG, HT29) were seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight under normoxia (21% O₂, 5% CO₂). Cells were then transferred to hypoxic conditions (1% O₂, 5% CO₂, 94% N₂) and treated with KC7F2 (0.1–10 μM) for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, and incubation continued for 4 h. DMSO (150 μL/well) was added to dissolve formazan crystals, and absorbance was measured at 570 nm. Cell viability (%) = (treated absorbance/control absorbance) × 100, and IC50 values were calculated using GraphPad Prism software [1]
- Western blot for HIF-1α and target proteins: Hypoxic HeLa/U87MG cells (1% O₂, 24 h) were treated with KC7F2 (1–5 μM), lysed in RIPA buffer (with protease inhibitors), and 30 μg of protein was separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk (1 h, room temperature), and incubated with primary antibodies (anti-HIF-1α, anti-VEGF, anti-cleaved PARP, anti-β-actin) overnight (4°C). HRP-conjugated secondary antibodies were added (1 h, room temperature), and bands were visualized via ECL. Band intensity was quantified using ImageJ software [1]
- qPCR for HIF-1α target genes: Hypoxic HeLa cells (1% O₂, 24 h) treated with KC7F2 (1–5 μM) were used for total RNA extraction. RNA was reverse-transcribed to cDNA, and qPCR was performed using primers for HIF-1α, VEGF, GLUT1, CAIX, and GAPDH (internal control). Relative mRNA levels were calculated using the 2⁻ΔΔCt method, and fold changes vs. vehicle control were reported [1]
- Annexin V-FITC/PI apoptosis assay: Hypoxic U87MG cells (1% O₂) were treated with KC7F2 (2–4 μM) for 48 h, harvested by trypsinization, washed with cold PBS, and resuspended in binding buffer. Annexin V-FITC (5 μL) and PI (10 μL) were added, and cells were incubated in the dark (15 min, room temperature). Apoptotic cells were analyzed via flow cytometry, and the percentage of early (Annexin V-positive/PI-negative) and late (Annexin V-positive/PI-positive) apoptotic cells was calculated [1]

U87MG glioblastoma xenograft protocol: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ U87MG cells (suspended in 100 μL of a 1:1 mixture of PBS and matrigel) into the right flank. When tumors reached an average volume of ~100 mm³, mice were randomly divided into two groups (n=6/group): (1) Vehicle group: 0.5% methylcellulose in PBS, administered via oral gavage once daily; (2) KC7F2 group: 15 mg/kg KC7F2 dissolved in 0.5% methylcellulose in PBS, administered via oral gavage once daily. Treatment was continued for 21 days. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2), and body weight was recorded weekly. At the end of treatment, mice were euthanized, and tumor tissues were excised for western blot analysis of HIF-1α and VEGF protein levels [1]

In vitro normal cytotoxicity: KC7F2 (≤10 μM) had no significant effect on cell viability (MTT assay, viability >90% vs. control group) in human normal foreskin fibroblasts (HFF) and peripheral blood mononuclear cells (PBMCs) after 72 hours of treatment under hypoxic or normoxic conditions [1] - In vivo acute toxicity: Female nude mice (n=3 per group) were treated with KC7F2 at doses of 10, 20 or 30 mg/kg (by gavage, once daily) for 7 days. No deaths or obvious toxic reactions (e.g., decreased activity, abnormal feeding) were observed. Mild weight loss (approximately 4%) occurred in the 30 mg/kg group, which returned to normal after drug withdrawal. No histopathological changes were found in the liver, kidneys and spleen [1]
- Plasma protein binding rate: The protein binding rate of KC7F2 in human plasma was 82%, as determined by balanced dialysis (37°C, 4 hours) [1]

[1]. Identification of a novel small molecule HIF-1alpha translation inhibitor. Clin Cancer Res. 2009 Oct 1;15(19):6128-6136.

Mechanism of action: KC7F2 specifically inhibits HIF-1α translation by targeting the 5' untranslated region (5' UTR) of HIF-1α mRNA, which is essential for cap-independent translation of HIF-1α under hypoxic conditions. KC7F2 does not affect HIF-1α transcription (no change in HIF-1α mRNA levels) or post-translational regulation (no effect on PHD2-mediated hydroxylation or VHL-dependent HIF-1α degradation) [1].
- Therapeutic potential: KC7F2 is a potential therapeutic agent for the treatment of cancers with high HIF-1α expression (e.g., glioblastoma, lung cancer, colorectal cancer) because HIF-1α drives tumor angiogenesis, glycolysis and metastasis. Its hypoxia-selective activity reduces toxicity to normal tissues (with lower HIF-1α levels) [1]
- Preclinical relevance: In the U87MG xenograft model, KC7F2 not only inhibited tumor growth but also reduced intratumoral angiogenesis by downregulating VEGF, which was observed by CD31 immunohistochemistry (tumor microvessel density was reduced by 58% compared to the vector group) [1]

C16H16CL4N2O4S4

570.38

567.874

927822-86-4

16047442

White to off-white solid powder

1.6±0.1 g/cm3

708.8±70.0 °C at 760 mmHg

382.5±35.7 °C

0.0±2.3 mmHg at 25°C

1.643

6.39

2

8

11

30

671

0

REQLACDIZMLXIC-UHFFFAOYSA-N

InChI=1S/C16H16Cl4N2O4S4/c17-11-1-3-13(19)15(9-11)29(23,24)21-5-7-27-28-8-6-22-30(25,26)16-10-12(18)2-4-14(16)20/h1-4,9-10,21-22H,5-8H2

2,5-dichloro-N-[2-[2-[(2,5-dichlorophenyl)sulfonylamino]ethyldisulfanyl]ethyl]benzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 32 mg/mL (~56.10 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.38 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)